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Biomedical subjects

Carl G Maki

Publications and source records attributed to Carl G Maki.

9 recordsLinked to original sources
PubMed2006

P53 and p73 differ in their ability to inhibit glucocorticoid receptor (GR) transcriptional activity.

BACKGROUND: p53 is a tumor suppressor and potent inhibitor of cell growth. P73 is highly similar to p53 at both the amino acid sequence and structural levels. Given their similarities, it is important to determine whether p53 and p73 function in similar or distinct pathways. There is abundant evidence for negative cross-talk between glucocorticoid receptor (GR) and p53. Neither physical nor functional interactions between GR and p73 have been reported. In this study, we examined the ability of p53 and p73 to interact with and inhibit GR transcriptional activity. RESULTS: We show that both p53 and p73 can bind GR, and that p53 and p73-mediated transcriptional activity is inhibited by GR co-expression. Wild-type p53 efficiently inhibited GR transcriptional activity in cells expressing both proteins. Surprisingly, however, p73 was either unable to efficiently inhibit GR, or increased GR activity slightly. To examine the basis for this difference, a series of p53:p73 chimeric proteins were generated in which corresponding regions of either protein have been swapped. Replacing N- and C-terminal sequences in p53 with the corresponding sequences from p73 prevented it from inhibiting GR. In contrast, replacing p73 N- and C-terminal sequences with the corresponding sequences from p53 allowed it to efficiently inhibit GR. Differences in GR inhibition were not related to differences in transcriptional activity of the p53:p73 chimeras or their ability to bind GR. CONCLUSION: Our results indicate that both N- and C-terminal regions of p53 and p73 contribute to their regulation of GR. The differential ability of p53 and p73 to inhibit GR is due, in part, to differences in their N-terminal and C-terminal sequences.

Animals↗
PubMed2005

Control of p53 nuclear accumulation in stressed cells.

Wild-type p53 accumulates in the nucleus following stress. Current models suggest this nuclear accumulation involves phosphorylation at p53 N-terminal sites, and inhibition of murine double minute (MDM)2-dependent nuclear export. We monitored the effects of stress on MDM2-dependent nuclear export of wild-type p53 and a mutant lacking N-terminal phosphorylation sites. Etoposide and ionizing radiation inhibited nuclear export of wild-type p53 and the phosphor-mutant to comparable extents, indicating nuclear export inhibition does not require N-terminal phosphorylation. Cytoplasmic p53 accumulated in the nucleus of transfected cells treated with the nuclear export-inhibitor leptomycin B (LMB). Interestingly, LMB caused less p53 nuclear accumulation than stress treatment, suggesting stress-induced nuclear accumulation of p53 does not result solely from inhibited nuclear export.

Active Transport, Cell Nucleus↗
PubMed2005

Cdk2-dependent Inhibition of p21 stability via a C-terminal cyclin-binding motif.

p21 is a member of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors that includes p21, p27, and p57. Recent studies have suggested that Cdk2 activity may promote p21 degradation through a pathway similar to that for p27, although the mechanism by which this occurs has not been clarified. In the current report, co-expression with cyclin E and Cdk2 stabilized p21 in a manner that required the CDK-binding site of p21 and a cyclin-binding site (cy1) located in the p21 N terminus. Strikingly, however, a kinase-dead Cdk2 mutant stabilized p21 to a greater extent than did wild-type Cdk2, consistent with the notion that Cdk2 activity can destabilize p21. The ability of wild-type Cdk2 to destabilize p21 required a potential Cdk2 phosphorylation site in p21 at serine 130 and an intact cyclin-binding motif (cy2) in the p21 C terminus. Finally, p21 was phosphorylated by Cdk2 at Ser-130 in vitro, and this ability of Cdk2 to phosphorylate p21 was dependent, in large part, on the presence of cy2. These results support a model in which active Cdk2 destabilizes p21 via the cy2 cyclin-binding motif and p21 phosphorylation.

Amino Acid Motifs↗
PubMed2004

A link between p73 transcriptional activity and p73 degradation.

The p53 family of proteins includes three members, p53, p63, and p73. The levels and stability of p53 are controlled in large part by MDM2, which can bind the p53 N-terminus and promote its degradation. Because the MDM2 gene is transcriptionally activated by p53, it forms part of an autoregulatory feedback loop that directly links the transcriptional activity of p53 with its degradation. In contrast, little is known about the mechanisms that control p63 or p73 stability. In the current study, p73 deletion or point mutants that lacked transactivation activity were stable compared to wild-type p73. A naturally occurring p73 variant (DeltaNp73) was also stable compared to wild-type p73. Finally, fusion of the VP16-transactivation domain to an inactive, stable p73 mutant restored transactivation function and rendered the mutant protein unstable. These results demonstrate that p73 transactivation activity is necessary for rapid p73 turnover, and suggest that one or more transcriptional targets of p73 may promote its degradation.

Animals↗
PubMed2003

MDM2 and promyelocytic leukemia antagonize each other through their direct interaction with p53.

p53 can be regulated through post-translational modifications and through interactions with positive and negative regulatory factors. MDM2 binding inhibits p53 and promotes its degradation by the proteasome, whereas promyelocytic leukemia (PML) activates p53 by recruiting it to multiprotein complexes termed PML-nuclear bodies. We reported previously an in vivo and in vitro interaction between PML and MDM2 that is independent of p53. In the current study, we investigated whether interaction between MDM2 and PML can indirectly affect p53 activity. Increasing amounts of MDM2 inhibited p53 activation by PML but could not inhibit PML-mediated activation of a p53 fusion protein that lacked the MDM2-binding domain. Conversely, increasing amounts of PML could overcome p53 inhibition by MDM2 but could not overcome MDM2-mediated inhibition of a p53 fusion protein that lacked the PML-binding domain. These results demonstrate that MDM2 and PML can antagonize each other through their direct interaction with p53 and suggest the combined effects of MDM2 and PML on p53 function are determined by the relative level of each protein. Furthermore, these results imply that interactions between MDM2 and PML by themselves have little or no effect on p53 activity.

Animals↗
PubMed2003

Physical and functional interactions between PML and MDM2.

The tumor suppressor protein PML and oncoprotein MDM2 have opposing effects on p53. PML stimulates p53 activity by recruiting it to nuclear foci termed PML nuclear bodies. In contrast, MDM2 inhibits p53 by promoting its degradation. To date, neither a physical nor functional relationship between PML and MDM2 has been described. In this study, we report an in vivo and in vitro interaction between PML and MDM2 which is independent of p53. Two separate regions of PML are recognized which can interact with MDM2. The C-terminal half of PML, encoded by residues 300-633, can interact with the central region of MDM2 which includes the MDM2 acidic domain. In addition, PML amino acids 1-200, which encode the RING-finger and most of the B box zinc binding motifs, can interact with the C-terminal, RING-finger containing region of MDM2. Interestingly, PML mutants in which sumoylation at lysine 160 was inhibited displayed an increased association with MDM2, suggesting that sumoylation at this site may be a determinant of PML-MDM2 binding. Coexpression with MDM2 caused a redistribution of PML from the nucleus to the cytoplasm, and this required the PML N terminus and the MDM2 RING-finger domain. These results suggest that interaction between the PML N terminus and MDM2 C terminus can promote PML nuclear exclusion. Wild-type MDM2 inhibited the ability of PML to stimulate the transcriptional activity of a GAL4-CBP fusion protein. This inhibition required the central, acidic region of MDM2, but did not require the MDM2 C terminus. Taken together, these studies demonstrate that MDM2 and PML can interact through at least two separate protein regions, and that these interactions can have specific effects on the activity and/or localization of PML.

Animals↗
PubMed2002

Nuclear import and export signals in control of the p53-related protein p73.

The p53-family of proteins, including p53, p63, and p73, shares a high degree of structural similarity and can carry out some redundant functions. However, mechanisms that regulate the localization and activity of these proteins have not been fully clarified. In this study, a nuclear localization signal (NLS) was identified in p73, which is required for p73 nuclear import and which could promote the nuclear import of a heterologous, cytoplasmic protein. Mutants lacking the NLS localized to the cytoplasm and displayed diminished transcriptional activity. A nuclear export signal (NES) was also recognized in p73s C terminus, the deletion of which caused p73 to display a more nuclear localization pattern. This NES was sensitive to leptomycin B and could function as an independent export signal when fused to a heterologous protein. Interestingly, p73 mutant proteins lacking the NLS or the NES were more stable than wild-type p73, suggesting that nuclear import and nuclear export are required for efficient p73 degradation. Our results indicate that p73 localization is controlled by both nuclear import and export and suggest that the overall distribution of p73 is likely to result from the balance between these two processes. Proper control of nuclear import and export is likely to be an important regulatory determinant of p73.

Animals↗